The present invention relates to methods and compositions for the treatment of xcex1-galactosidase A deficiency.
Fabry disease is an X-linked inherited lysosomal storage disease characterized by severe renal impairment, angiokeratomas, and cardiovascular abnormalities, including ventricular enlargement and mitral valve insufficiency. Fabry disease also affects the peripheral nervous system, causing episodes of agonizing, burning pain in the extremities. Fabry disease is caused by a deficiency in the enzyme xcex1-galactosidase A (xcex1-Gal A). xcex1-Gal A is the lysosomal glycohydrolase that cleaves the terminal xcex1-galactosyl moieties of various glycoconjugates. Fabry disease results in a blockage of the catabolism of the neutral glycosphingolipid, ceramide trihexoside (CTH), and accumulation of this enzyme substrate within cells and in the bloodstream.
Due to the X-linked inheritance pattern of the disease, most Fabry disease patients are male. Although severely affected female heterozygotes have been observed, female heterozygotes are often asymptomatic or have relatively mild symptoms (such as a characteristic opacity of the cornea). An atypical variant of Fabry disease, exhibiting low residual xcex1-Gal A activity and either very mild symptoms or apparently no other symptoms characteristic of Fabry disease, correlates with left ventricular hypertrophy and cardiac disease. Nakano et al., New Engl. J. Med. 333: 288-293 (1995). A reduction in xcex1-Gal A may be the cause of such cardiac abnormalities.
The cDNA and gene encoding human xcex1-Gal A have been isolated and sequenced. Human xcex1-Gal A is expressed as a 429-amino acid polypeptide, of which the N-terminal 31 amino acids are the signal peptide. The human enzyme has been expressed in Chinese Hamster Ovary (CHO) cells (Desnick et al., U.S. Pat. No. 5,356,804; Ioannou et al., J. Cell Biol. 119: 1137 (1992)); and insect cells (Calhoun et al., WO 90/11353).
However, current preparations of xcex1-Gal A have limited efficacy. Methods for the preparation of xcex1-Gal A with relatively high purity depend on the use of affinity chromatography, using a combination of lectin affinity chromatography (concanavalin A (Con A) Sepharose(copyright)) and affinity chromatography based on binding of xcex1-Gal A to the substrate analog N-6-aininohexanoyl-xcex1-D-galactosylamine coupled to a Sepharose(copyright) matrix. See, e.g., Bishop et al., J. Biol. Chem. 256: 1307-1316 (1981). The use of proteinaceous lectin affinity resins and substrate analog resins is typically associated with the continuous leaching of the affinity agent from the solid support (Marikar et al., Anal. Biochem. 201: 306-310 (1992), resulting in contamination of the purified product with the affinity agent either free in solution or bound to eluted protein. Such contaminants make the product unsuitable for use in pharmaceutical preparations. Bound substrate analogs and lectins can also have substantial negative effects on the enzymatic, functional, and structural properties of proteins. Moreover, xcex1-Gal A produced by the methods in the prior art is rapidly eliminated by the liver.
Thus, a need remains in the art for a purification protocol using conventional chromatography resins, which are readily available in supplies and quality suitable for large-scale commercial use, and which produces an xcex1-Gal A preparation that is free of affinity agent. In addition, a need remains in the art for xcex1-Gal A preparations with an increased circulating half-life and increased uptake in specific tissues other than liver.
The invention provides highly purified xcex1-Gal A preparations, and various methods for purifying the xcex1-Gal A glycoforms. The invention also provides xcex1-Gal A preparations with altered charge and methods for making those preparations. Charge alterations are achieved by increasing the sialic acid content of xcex1-Gal A and/or by increasing the phosphorylation of xcex1-Gal A. The invention further provides xcex1-Gal A preparations that have an extended circulating half-life in a mammalian host, and methods for making same. Finally, the present invention further provides methods and dosages for administering an xcex1-Gal A preparation to a subject. The xcex1-Gal A preparations of the present invention will be useful for treatment of individuals with Fabry disease or atypical variants of Fabry disease, e.g., specific populations of Fabry patients with predominantly cardiovascular abnormalities, such as ventricular enlargement, e.g., left ventricular hypertrophy (LVH), and/or mitral valve insufficiency, or Fabry patients with predominantly renal involvement.